An Integrated Approach Based on Multiplexed Protein Array and iTRAQ Labeling for In-Depth Identification of Pathways Associated to IVF Outcome

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.高通量蛋白质定量技术的问世，使得研究人员可通过纵向与横断面研究，对生理及病理状态下的生物体液开展全面表征。尤为关键的是，借助整合式多重分析手段同步探究细胞因子、生长因子信号通路及其相关下游效应分子，可为解析生命系统中的生物网络与生命过程提供强有力的研究策略。越来越多的研究表明，人类生殖液中的生物活性分子——包括人类卵泡液（human follicular fluid, hFF）——可影响卵母细胞质量、受精过程与胚胎发育，进而可能对妊娠相关病症的病理生理进程产生影响。本研究采用同位素标记相对和绝对定量（iTRAQ）标记策略结合多重蛋白芯片技术对人类卵泡液进行分析，旨在探究与体外受精（in vitro fertilization, IVF）结局相关的生物学过程与通路。该iTRAQ标记策略实现了对89种蛋白质的定量分析，其中30种蛋白质在IVF结局成功与失败的人类卵泡液样本中存在差异表达。本靶向研究依托多重抗体蛋白芯片技术，可同时定量27种低丰度蛋白质，涵盖具备促炎与抗炎活性的生长因子、趋化因子及细胞因子。大量差异调控的蛋白质参与了凝血、急性期反应信号通路及补体系统相关的生物学功能。综上，本研究结果全面阐明了与IVF结局相关的人类卵泡液蛋白质组变化，进而深化了生殖医学与生殖研究领域的现有认知。